A complete overview of commercially available biocontrol agents can be found in ‘The Manual of Biocontrol Agents, Fourth Edition’, edited by Copping in 2004 [7]. However, within the frame of this dissertation I mainly focused on indirect PGP against two severe potato pathogens Rhizoctonia solani and Phytophthora infestans. Although the focus within this thesis was mainly on PGP bacteria, arbuscular mycorrhizal fungi (AMF) are also known to suppress R. solani and P. infestans induced diseases in plants. R. solani is the causing agent of scurf disease and stem canker in potato plants [28]. P.
infestans causes potato late blight disease, one of the most devastating diseases of potato worldwide
[29,30]. R. solani is difficult to control because it has the ability to survive as sclerotia under adverse environmental conditions for many years, is capable of surviving as a saprophyte and has a very wide host range [31]. Disease transmission occurs via infected seed tubers. P. infestans infection, on the other hand, mainly occurs from airborne contamination by sporangia [32]. These sporangia can spread over wide distances during the potato growing season. P. infestans infections are very aggressive and are often associated with complete field destruction. Moreover, the time required for the pathogen to complete its life-cycle can be as short as three days, and as such thousands of spores can be formed in a very short period of time [33], contributing to the large scale often associated with infection.
20 1.3.1 Rhizoctonia solani disease suppression
R. solani disease occurs in potato production throughout the world [28]. Symptoms manifest on below-
and aboveground parts of the plant as black scurf and stem canker respectively [28] (Fig. 1.1).
Figure 1.1 Black scurf disease in potato, caused by Rhizoctonia solani infection. Extracted from [34].
Black scurf develops later in the growing season and can be recognized from the appearance of black, irregular sclerotia on the tuber. Although differences in susceptibility amongst potato cultivars have been observed, no resistant cultivars have been identified nor developed [28]. The species R. solani consists of a number of anastomosis groups (AGs) [35] that are not equally infective to potato. Currently, disease control occurs by chemical fungicides. However, the different AGs are not equally susceptible to these agents. Efficacy of disease control depends on the stage of infection, and whether the infection was soil borne or tuber borne. Tuber borne R. solani infections are relatively easy to control compared to soil borne infections [28]. Chemical fungicide treatments may not always be effective against soil borne infections. Treatments seem to perform well in the early stages of disease development; however, in the presence of high inoculum levels higher doses are needed to be effective. As a result, R. solani disease is a complex disease to manage. Various biological agents, however, have proven to have promising effects with respect to control of the pathogen. Literature study shows the
21
effectiveness of fungi belonging to the genera Trichoderma [36,37,38,39], Verticillium [40,41],
Cladorrhinum [42], binucleate Rhizoctonia [43], Streptomyces [44], and Gliocladium [45]. Likewise,
several members of bacterial genera have proven to be effective suppressors of R. solani disease. These include Bacillus [46,47], Burkholderia [47,48], and Pseudomonas [46,47,48]. Van den Boogert and Luttikholt (2004) [41] found that the biocontrol fungus Verticillium biguttatum had a synergistic effect on Rhizoctonia-specific (pencycuron, flutanolil) fungicides. They also found that V. biguttatum extended the control spectrum of oomycete-specific chemical fungicides (cymoxanil and propamocarb). Grosch et al. (2005) [49] found two Pseudomonas strains and one Serratia strain, all of which were isolated from potato roots, that were able to suppress R. solani disease during field trials with potato. Ikeda and colleagues (2012) [50] performed field tests with infected potato seed tubers to test the biocontrol efficacy of Pythium oligandrum and obtained disease suppression at a level similar to that achieved by chemical control. Moreover, their study showed the expression of defense-related genes in the potato plant, which reduced tuber disease severity upon challenge with R. solani. Their observations indicated that P. oligandrum induced resistance in the potato plant. Wilson et al. (2008) [36] performed field trials to test the efficacy of Trichoderma harzianum in controlling soil borne potato infection and found that T.
harzianum was capable of suppressing disease both in combination with the chemical fungicide
flutolanil and when applied alone. Disease suppression in both cases was higher than if flutolanil was used alone.
22 1.3.2 Phytophthora infestans disease suppression
P. infestans disease causes enormous economic damage, which is estimated at $5.2 billion worldwide
annually [51]. Late blight disease (Fig. 1.2) was responsible for the Great Famine in Ireland around 1845.
Figure 1.2 Phytophthora infestans infection in potato plants (Late blight disease). (A) Aboveground symptoms, (B)
belowground symptoms. Extracted from [52].
The worldwide breeding for resistant potato varieties only had little effect so far [51]. Frequently a genetic variety was obtained which seemed promising with respect to P. infestans resistance. However, whenever the variety was grown for a few years and at a larger scale, the resistance was repeatedly lost. Current disease control measurements consist of an array of tactics [32]. These include planting healthy seed tubers, eliminating on-farm sources that may be or become contaminated with P. infestans (e.g. destruction of potatoes in waste heaps), applying chemical fungicides for disease control [53] and elongating the time between potato planting cycles by means of crop rotation, which is necessary since
P. infestans survives in the soil after the growing season has ended [32]. Sexual reproduction of the
pathogen has created more aggressive P. infestans strains with increased virulence [52,53,54], thus increasing the need for pesticide application. However, increasing fungicide resistance in the pathogen populations simultaneously renders agrochemicals less effective. Moreover, fungicides to control late blight disease are based on copper, which is known to have a negative environmental impact [32]. Excess amounts of copper in the environment are harmful for aquatic and soil organisms [55], and may cause adverse health effects in humans [56]. In Belgium, over 1000 tons of active agents are applied annually to ensure control of P. infestans. In Flanders, an average of about 17 kg of active component is applied per hectare per year [52]. Therefore, public concern puts further pressure on the use of copper based fungicides. It is clear that there is a great need for alternative treatments. Genetic modification of potato varieties [52] or biopesticide applications may be valuable alternatives. However, due to public concern about genetically modified organisms, biopesticides may be the preferred approach.
23
Axel et al. (2012) [32] made an interesting overview of all published studies in which the biocontrol efficiency of microorganisms against P. infestans was tested. The authors concluded that so far the application of microorganisms as biological control agents did not result in any consistent field performance. However, more recently Wharton et al. (2012) [57] tested the efficiency of formulations of
Trichoderma harzianum and Bacillus subtilis in suppressing P. infestans in field trials with potato plants
and found that the B. subtilis formulations were able to control seed piece decay caused by P. infestans with 57% in trials performed in 2006 and with 98% in 2007. T. harzianum was able to suppress seed piece decay with 81.5% in 2006 and 77% in 2007. This was similar to the level of suppression obtained with a commercially available mixture of fludioxonil and mancozeb. The authors found that pre-storage conditions of treated tubers played a significant role in disease suppression activity, as sub-optimal storage conditions did not result in disease suppression. Similarly, disease emergence was higher with the chemical fungicide mixture after sub-optimal pre-storage conditions. The authors also noted the fact that due to the effective root colonization of T. harzianum, biocontrol applications were less prone to being washed away during the growing season, resulting in longer efficiency compared to chemical fungicides. Field trials that were performed by Dorn et al. (2007) [58] with a selection of commercially available biocontrol agents were less promising. None of the agents reached the same level of control as copper based fungicides. The failure to suppress late blight disease was mainly attributed to detrimental environmental conditions. The copper based fungicides were more stable. Although fungicide stability is a desired trait, it simultaneously raises concern about the persistence of copper-based preparations in the environment. Dorn et al. (2007) [58] did not perform pre-storage of treated tubers but sprayed the biopesticides onto already planted tubers which were subsequently infected with P. infestans a few weeks later. As Wharton et al. (2012) [57] demonstrated the importance of pre-storage, the results may have been better if Dorn et al. (2012) [58] had accounted for this. Axel and colleagues (2012) [32] postulated that direct application of metabolites responsible for P. infestans inhibition may be a valuable alternative for the application of the producing organisms. However as this is beyond the scope of this dissertation, I refer to their review for further information.